1. Field of the Invention
The present invention relates to the field of molecular biology and provides improved methods for the replication and amplification of ribonucleic acid (RNA) sequences. In a preferred embodiment, the invention provides a method for synthesizing a complementary DNA copy from an RNA template with a thermoactive DNA polymerase. In another aspect, the invention provides methods for coupling reverse transcription of an RNA template and amplification of the resultant DNA using a thermostable DNA polymerase. In a preferred embodiment RNA is reverse transcribed and amplified in a homogeneous, one tube, one enzyme reaction. Methods for sterilization of reverse transcription and reverse transcription/amplification reactions are also provided.
2. Description of Related Art
The term "reverse transcriptase" describes a class of polymerase characterized as RNA-dependent DNA polymerases. All known reverse transcriptases require a primer to synthesize a DNA transcript from an RNA template. Historically, reverse transcriptase has been used primarily to transcribe mRNA into cDNA which can then be cloned into a vector for further manipulation.
Avian myoblastosis virus (AMV) reverse transcriptase was the first widely used RNA-dependent DNA polymerase (Verma, 1977, Biochem.Biophys.Acta 473:1). The enzyme has 5'-3' RNA-directed DNA polymerase activity, 5'-3' DNA-directed DNA polymerase activity, and RNase H activity. RNase H is a processive 5' and 3' ribonuclease specific for the RNA strand of RNA-DNA hybrids (Perbal, 1984, A Practical Guide to Molecular Cloning, Wiley & Sons New York). Errors in transcription cannot be corrected by reverse transcriptase because known viral reverse transcriptases lack the 3'.fwdarw.5' exonuclease activity necessary for proofreading (Saunders and Saunders, 1987, Microbial Genetics Applied to Biotechnology, Croom Helm, London). A detailed study of the activity of AMV reverse transcriptase and its associated RNase H activity has been presented by Berger et al., 1983, Biochemistry 22:2365-2372.
Berger et al. found that the rate limiting step in the reverse transcription of RNA was initiation by the enzyme, rather than the sequential polymerization of additional nucleotides. To overcome this limitation, use of stoichiometric, rather than catalytic, quantities of reverse transcriptase is frequently recommended (Buell et al., 1978, J. Biol. Chem. 253:2471-2482; Wickens et al, 1978, Bio. Chem. 253:2483-2495; Yoo et al., 1982, Proc. Nat. Acad. Sci. USA 80:1194-1198; and Okayama and Berg, 1982, Mol. Cell. Biol. 2:161-170). However, when stoichiometric amounts of reverse transcriptase are used, the low level of RNase H activity is significant and may be responsible for fragmented cDNAs and limited cDNA yields (Kotewicz et al., 1988, Nuc. Acid Res. 16:265-277). Christopher et al., 1980, Eur. J. Biochem. 111:4190-4231, and Michelson et al., 1983, Proc. Nat. Acad. Sci. USA 80:472-476, have suggested that including an RNase inhibitor in cDNA reactions could alleviate this problem.
DNA polymerases isolated from mesophilic microorganisms such as E. coli. have been extensively researched (see, for example, Bessman et al., 1957, J. Biol. Chem. 233:171-177 and Buttin and Kornberg, 1966, J. Biol. Chem. 241:5419-5427). E. coli DNA polymerase I (Pol I) is useful for a number of applications including: nick-translation reactions, DNA sequencing, in vitro mutagenesis, second strand cDNA synthesis, polymerase chain reactions (PCR), and blunt end formation for linker ligation (Maniatis et al., 1982, Molecular Cloning: A Laboratory Manual Cold Spring Harbor, N.Y.).
Several laboratories have shown that some DNA polymerases are capable of in vitro reverse transcription of RNA (Karkas, 1973, Proc. Nat. Acad. Sci. USA 70:3834-3838; Gulati et al., 1974, Proc. Nat. Acad. Sci. USA 71:1035-1039; and Wittig and Wittig, 1978, Nuc. Acid Res. 5:1176-1178). Gulati et al. found that E. coli Pol I could be used to transcribe Q.beta. viral RNA using oligo(dT).sub.10 as a primer. Wittig and Wittig have shown that E. coli Pol I can be used to reverse transcribe tRNA that has been enzymatically elongated with oligo(dA). However, as Gulati et al. demonstrated, the amount of enzyme required and the small size of the cDNA product suggests that the reverse transcriptase activity of E. coli Pol I has little practical value.
The use of thermostable enzymes to amplify existing nucleic acid sequences in amounts that are large compared to the amount initially present was described in U.S. Pat. Nos. 4,683,195 and 4,683,202, which describe the polymerase chain reaction (PCR) processes. These patents are incorporated herein by reference. Primers, template, nucleoside triphosphates, the appropriate buffer and reaction conditions, and a polymerase are used in the PCR process, which involves denaturation of target DNA, hybridization of primers, and synthesis of complementary strands. The extension product of each primer becomes a template for the production of the desired nucleic acid sequence. These patents and U.S. Pat. No. 4,965,188, incorporated herein by reference, disclose that, if the polymerase employed is a thermostable enzyme, then polymerase need not be added after every denaturation step, because heat will not destroy the polymerase activity.
Thermostable DNA polymerases are not permanently inactivated even when heated to 93.degree.-95.degree. C. for brief periods of time, as, for example, in the practice of DNA amplification by PCR. In contrast, at this elevated temperature E. coli DNA Pol I and previously described reverse transcriptases are inactivated.
The thermostable DNA polymerase from Thermus aquaticus (Taq) has been cloned, expressed, and purified from recombinant cells as described in Lawyer et al., 1993, PCR Methods and Applications, 2:275-287; Lawyer et al., 1989, J. Biol. Chem. 264:6427-6437; and U.S. Pat. Nos. 4,889,818 and 5,079,352, which are incorporated herein by reference. Crude preparations of a DNA polymerase activity isolated from T. aquaticus have been described by others (Chien et al., 1976, J. Bacteriol. 127:1550-1557, and Kaledin et al., 1980, Biokymiya 45:644-651).
The thermostable DNA polymerase from Thermus thermophilus (Tth) has also been purified and is described in PCT Patent Publication No. WO91/09950, which is incorporated herein by reference. PCT Patent Publication No. WO91/09950 also describes that the gene encoding Tth DNA polymerase enzyme from Thermus thermophilus has been identified and cloned. Recombinant Tth provides an alternative means for preparing the thermostable enzyme. Crude preparations of DNA polymerase activity isolated from T. thermophilus have been described by Ruttimann et al., 1985, Eur. J. Biochem. 149:41-46. The thermostable DNA polymerase from Thermotoga maritima has been identified and cloned and is described in copending Ser. No. 567,244, filed Aug. 13, 1990, and PCT Patent Publication No. WO92/03556, which are incorporated herein by reference.
PCR requires a nucleic acid template and appropriate primers for amplification. The DNA to be amplified may be synthetic or genomic, contained in a plasmid, or contained in a heterogeneous sample. If the nucleotide sequence to be amplified is RNA, the nucleic acid molecule is first treated with reverse transcriptase in the presence of a primer to provide a cDNA template for amplification. Prior to the present invention, amplification of RNA necessitated a reverse transcription step with, e.g., a non-thermostable reverse transcriptase such as Molony Murine Leukemia Virus Reverse Transcriptase (MoMuLV RT) or AMV-RT, followed by treatment of the resulting single-stranded cDNA with a DNA polymerase. The amplification of RNA could be greatly simplified by the availability of a method for reverse transcribing RNA and amplifying DNA with a single enzyme.
Taq polymerase has been reported to inefficiently synthesize cDNA using Mg.sup.+2 as the divalent metal ion (Jones and Foulkes, 1989, Nuc. Acids. Res. 176:8387-8388). Tse and Forget, 1990, Gene 88:293-296; and Shaffer et al., 1990, Anal. Biochem. 190:292-296, have described methods for amplifying RNA using Taq polymerase and Mg.sup.+2 ion. However, the methods are inefficient and insensitive. For example, Tse and Forget demonstrate that 4 .mu.g of total RNA is required to generate sufficient PCR product for ethidium bromide-stained gel visualization, using an abundantly expressed mRNA target. In addition, false positive signals from DNA template (PCR product from prior reactions or plasmid DNA contamination) were not rigorously excluded.
The present invention addresses this need and provides high temperature cDNA synthesis by thermoactive DNA polymerases. The present invention provides improved methods for a one enzyme, one tube, coupled reverse transcription/amplification assay using a thermostable DNA polymerase. The need to open the reaction vessel and adjust reaction components between the two steps is eliminated. The methods offer enhanced sensitivity, simplicity, and specificity over current methods.